Our laboratory studies the mechanism of action of the 70 kDa class of heat shock proteins (Hsp70s), which have been termed molecular chaperones because they are involved in the folding and unfolding of proteins and in the formation and dissociation of protein complexes. In these studies we have concentrated on exploring the role of Hsp70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. In many of their activities the Hsp70s require cofactors known as J-domain proteins that induce protein substrates to bind to Hsp70, and we previously discovered that uncoating also requires a J-domain protein, the minor 100 kDa clathrin assembly protein (AP), auxilin. Auxilin is a nerve specific protein and we later discovered that the non-neuronal homolog of auxilin is the 150 kDa protein GAK (auxilin2). We then showed that C. elegans has a single gene for auxilin and when auxilin expression is inhibited by RNA-mediated interference, there is a marked inhibition of clathrin-mediated endocytosis which in turn causes the worms to arrest during larval development. We also showed that yeast has a single gene for auxilin and that when this gene is deleted the resulting haploid yeast mutants showed an increase of clathrin associated with vesicles and a corresponding decrease in free clathrin in the cytosol. In addition, there was a marked decrease in transport of both carboxypeptidase Y and the G-protein-coupled receptor Ste3 to the vacuole; transport that normally occurs through clathrin-mediated endocytosis. From these data, we concluded that Hsp70 and auxilin are required for a fundamental step in clathrin-mediated endocytosis. Last year we investigated whether this fundamental step might not only involve dissociation of clathrin after clathrin-coated vesicles but also dissociation and rebinding of clathrin, i.e. clathrin exchange, occurring during invagination of the clathrin-coated pit. By blocking clathrin-mediated endocytosis under conditions where clathrin-coated pits on the plasma membrane remain intact, we demonstrated that in the presence of ATP clathrin in clathrin-coated pits exchanges with free clathrin in the cytosol. We also found that AP2 in the clathrin-coated pits exchanged with free AP2 and this exchange occurred at about the same rate as clathrin exchange. Likewise, at 15 ?C where transport out of the trans-Golgi network is blocked, both clathrin and AP1 exchanged with free clathrin and AP1 in the cytosol, respectively. From these data we concluded that clathrin-coated pits at both the plasma membrane and the trans-Golgi network are dynamic structures that show rapid exchange of both clathrin and APs. Furthermore, we concluded that the ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits and therefore may be involved in the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. During the past year we investigated whether Hsc70 and auxilin are involved in the clathrin exchange that occurs during clathrin-mediated endocytosis. We approached this question by first investigating whether clathrin and AP2 can be induced to dissociate from permeablized cells. We found that, while Hsp70 and ATP alone could not dissociate the clathrin from the clathrin-coated pits on the plasma membrane of permeablized cells, the addition of auxilin caused rapid uncoating of the clathrin-coated pits. Clathrin present on the trans-Golgi network was also uncoated by Hsc70 and auxilin in these cells. However, this uncoating was specific for clathrin; the AP2 present on the pits was not removed. Furthermore, even when clathrin was added along with Hsp70 and auxilin, we could not detect clathrin exchange. In contrast, when cytosol was added to the permeablized cells AP2 as well as clathrin was removed from the clathrin-coated pits, and furthermore this dissociated clathrin and AP2 was replaced on the same pits with free clathrin and AP2 from the cytosol. To show that this exchange required Hsc70, we showed that J-domain proteins that inhibit uncoating in vitro markedly reduces both the exchange of clathrin and AP2. From these data we have concluded that the combination of Hsc70 and auxilin can uncoat clathrin-coated pits as well as clathrin-coated vesicles in an ATP dependent reaction in permeablized cells. This is consistent with the view that Hsc70 and auxilin are required for the clathrin-exchange that occurs on clathrin-coated pits in vivo. In addition to these studies on permeablized cells we have continued our studies on both the auxilin and GAK knock-out mice. We have found that the GAK knock-out is lethal before implantation of the embryo. Therefore, we are now making a CRE-LOX knockout so that we can test the effect of the GAK knock-out after development of the embryo. In regard to the auxilin knock-out we have found that here some homozygous mice do develop to maturity and seem normal. But interestingly the homozygous mice seem unable to mate with each other and in addition the number of homozygous mice born in heterozygous-homozygous crosses seems reduced by about 50%. This suggest that the some of the homozygous auxilin knock-out mice are dying during embryonic development. Characterizing both the GAK and auxilin knock-out mice will be a major effort of the laboratory during the coming year.